Upregulation Of Orexin Receptor 1 In PVN Contributed To Hypertension Of Obesity Zucker Rats

The obesity morbidity has been increasing world widely, and its potential risks relate to kinds of healthy problem including hypertension, stroke, heart attack and infraction. Especially obesity was considered as an important pathophysiological contributor to hypertension development. In 1998, two independent research groups almost simultaneously discovered one neuron peptide-orexin, also called hypocretin. Orexin is one excitatory neuropeptide synthesized in lateral and posterior hypothalamus. Two subtypes of orexin are found, orexin A(OXA) and orexin(OXB). Both orexin peptides compete to interact with two G-protein coupled receptors, orexin receptor 1(OXR1) and orexin receptor 2(OXR2) with different binding affinities. OXA approximately equally binds to the two receptors, while OXB mainly binds to OXR2 with 5 times greater potent than OXR1.Although orexin neurons only concentrated in the lateral and posterior hypothalamus, its axons extends to almost whole brain area. Orexins were initially revealed to regulate appetite and energy balance based on that intracereventrical orexin microinjection increased food intake and energy expenditure. Additionally, orexin mutation or orexin knockout animals developed sleep disorders canine narcolepsy, implicating its role in sleep regulation. Moreover, orexin receptors are widely distributed in cardiovascular nucleus, such rostral ventralateral medulla(RVLM), NTS, locus coeruleus(LC), lateral paragigantocellular nucleus, midbrain periaqueductal gray and even in intermediolateral cell column of spinal cord and sympathetic pre-ganglionic neurons. The resting blood pressure decreased in both orexin-deficient rats and mice by genetically ablating orexin neurons, suggesting a necessary role of orexin keeps hemostasis of hemodynamics. Animal studies in vivo found that microinjection of orexins or antagonists into several cardiovascular nucleus caused obvious pressor response. Anesthetized and conscious normotensive animals showed higher blood pressure and enhanced sympathetic outflow, accompanied with an increase of plasma catecholamines following intracereventrol microinjection of orexin A or orexin B. The orexin-induced pressor effects could be abolished by chemical blockade of ganglion or orexin receptors. These studies provided evidence that central overactivation of orexin system would misbalance autonomic control, thus enhancing sympathetic outflow to elevate blood pressure. In vitro, through patch clamp technique much studies looked into neuronal electrophysiological responses to orexins or antagonist application. Orexins depolarized membrane potential of RVLM and NTS neurons and does-dependently increased spontaneous firing frequency. These orexin-excitatory effects were attenuated with antagonist pretreatment against orexin receptors. It has been found that orexins promoted presynaptic release of glutamate, thus increasing excitatory presynaptic currents in lateral hypothalamus neurons. Moreover, rather than presynaptic excitatory effects, orexins application elevated celluar calcium influx through L-type calcium channel open due to activation by G-protein signal transduction. In addition, it was found that the postsynaptic non-selective canton channel could be strongly activated around resting membrane potential by orexin. Overall, orexins elicits excitatory influences on neuronal electrophysiological activities through distinct mechanism depending on neurons types.The paraventricular nucleus of hypothalamus(PVN), an important central nucleus composed of distinct functional neuron groups. PVN serves as a bridge to participate in neuron-endocrine response, thus promoting release of several hormones under physical conditions. The connectional existence has been determined from PVN projecting to RVLM, NTS and the spinal cord. Especially, sympathomotor axons of PVN directly project to theoretic presympathetic neurons in spinal cord. These spinal projections have been widely determined to enhance output of sympathetic tone. In the spontaneous hypertensive rats, blockade over-excitation from these presympathetic neurons, relative to the wild type improved the higher artery pressure due to attenuation of peripheral sympathetic activity.The obesity Zucker rats share common pathophysiological features with human being of obesity, such as higher blood pressure, sympathetic overactivation and higher norepinephrine as previous studies reported. This obesity model of hypertension has been widely used to look into mechanisms underlying hypertension development relating with obesity. Therefore, in the present study we observed pressor influences of orexin receptors in PVN on peripheral blood pressure in obesity Zucker rats. In addition, we tested the presympathetic neuronal responses to orexin and/or antagonist application and explored the underlying mechanisms. Finally, we observed whether downregulation of orexin receptor 1 in PVN by OXR1-sh RNA could decrease sympathetic outflow and norepinephrine level, thus attenuating the blood pressure.Part one: upregulation of PVN orexin receptor contributes to hypertension in obesity Zucker rats.Purpose: Over-activation of sympathetic tone underlies one main neurophysiological contributor in obesity-associated hypertension. As an important endocrine nucleus, paraventricular nucleus of hypothalamus also could directly spinally project to the theoretic sympathetic afferent system, thus regulating autonomic balance. Excitatory peptide of orexin and its receptors has been widely shown to cause pressor influence in normotensive and hypertensive animals. Thus, this part explored the possible pressor effects of PVN orexin receptors in Zucker obesity rats(OZRs), one widely used model for hypertension research.Methods:First, PVN microinjection experiments were performed to look into effects of orexin A and antagonists of its receptors on artery pressure, heart rate and renal sympathetic activity in anesthetized adult Zucker rats. Additionally, western blot was used to compare different changes of PVN orexin receptors in lean and obesity Zucker rats. To further confirm the over-activation of orexin receptors causally related with hypertension development, we also measured expression of PVN orexin receptors in 6-week rats of each phenotype.Results:Under anesthetization, the basal MAP of OZRs rats was higher than the LZRs. Microinjection of SB33487, one selective antagonist for orexin receptor 1 into PVN significantly decreased blood pressure, heart rate and RSNA in OZRs, compared with the baseline, and however, no changes were found in LZRs. For the selective OXR2 antagonist, TCS209 PVN microinjection caused no influences on hemodynamics on either OZRs or LZRs. To confirmpressor effects of PVN OXR1, we injected orexin A with more affinities to OXR1 than OXR2, and found that an obvious increase of MAP and RNNA were induced after orexin A injection, and these were blocked by pre-treated with SB334867, not TCS209. Consistent with these functional results, we also found a significant upregulation of PVN OXR1 in OZRs, compared with LZRs, but not changes for OXR2. Although body weight in 6-week-old OZRs was much higher than LZRs, the artery blood pressure and PVN OXR1 did not show significant difference between the phenotypes.Summary:Collectively, these data suggested that OXR1 overexpression in PVN caused blood pressure increase, relating with more output from sympathetic tone output in OZRs.Part two: overactivation of orexin receptor 1 caused hyperexcitability of presympathetic neurons in PVN of obesity Zucker rats.Purpose: The paraventricular nucleuses of hypothalamus not only participates in neuron-endocrine responses, but were also revealed to regulate sympathetic tone throughdirect projections to the latermedial spinal column. The hyperactivity of pre-sympathetic neurons in PVN accounts for hypertension development in spontaneous hypertensive rats. Orexin has been observed to elicit depolarization of neurons in several cardiovascular nucleus such RVLM and NTS through OXR1 and/or OXR2 signaling. Based on the findings of first part that orexin A injected to PVN resulted stronger pressor effects in OZRs than LZRs by upregulation of OXR1. Therefore, the present part furtherly observed that whether overactivation of OXR1 contributed to hyper-activities of pre-sympathetic neurons in PVN and explored the underlying mechanisms.Method:(1) The presympathetic neurons in PVN were retrogradely identified by red Fluo Sphere injected into the latermedial spinalcolumn at theoretic levels;(2) The membrane potential and firing activity of retrogradely-labeled neurons were observed and recorded under current clamp of whole cell;(3) To identify the retrogradely-labeled neurons expressed OXR1, immunostaing was performed against OXR1 in PVN and confocal images were captured and analyzed by Image J software.Results:(1) The basal membrane potential and firing frequency of presympathetic neurons were significantly higherin OZRs than LZRs;(2) Orexin A application excited these neurons, with more increment of spontaneous activities in OZRs compared with LZRs;(3) These orexin-induced excitatory effects were blocked by OXR1 selective inhibitor SB334867, but not by OXR2 inhibitor TCS209;(4) Confocal images observed that over 95% percentage of presympathetic neurons contained OXR1, with no difference between two phenotypes.(5) Orexin A application did not influence excitatory presynaptic currents EPSC;(6) Non-selective canton current induced by orexin A were larger at the hypolarization level in OZRs compared to LZRs.Summary: This part looked into the neuronal mechanism accounting for orexin-induced hyperactivities in the presympathetic neurons in OZRs. It found that OXR1, not OXR2 caused depolarization of the presympathetic neurons through increasing postsynaptic NSCC in OZRs.Third part: downregulation of PVN OXR1 decreased artery pressure by attenuation sympathetic output in obesity Zucker rats.Purpose: Orexin, one excitatory peptide synthetized in the lateral hypothalamus has been shown to depolarize neurons in cardiovascular-regulated nucleus. In vivo studies found that orexin microinjection to RVLM and NTS increased blood pressure, heart rate and also sympathetic activities. In the above two pars, we found that overactivation of OXR1 signaling of presympathetic neurons in PVN resulted in hypertension in OZRs by shifting autonomic nervous system. Therefore in the last part, by using si RNA technique we explored whether downregulation of OXR1 in PVN could reverse the hypertension in OZRs.Method:(1) The OXR1-sh RNA were designed by company and GFP gene was inserted and promoted when OXR1-si RNA transcripted, and as control virus, scrambled virus carried nonfunctional RNA;(2) The wireless telemetry continuously monitored the artery pressure in vivo for 42 days by Powerlab after OXR1-sh RNA injected to PVN. The pulse-pulse intervals(PI) were distinguished and extracted from the pulse tracing using Labchart 8.0; PI were then analyzed and transformed to frequency domain of heart rate variability composing of three main domains: very low frequency(v LF), low frequency(LF) and high frequency(HF). Ration of LF/HF was calculated to reflect sympathetic system function;(3) Additionally, OXR1 expression was qualified using western bot, and for further morphological identification, immunostaing was performed to compare OXR1 distribution between two phenotypes;(4) We also injected orexin A and antagonists of orexin receptors into PVN and observed influences on renal sympathetic activities;(5)Finally, in vitro, firing activity of presympathetic neurons was observed under current clamp of whole cell.Results:(1) At the baseline no differences of MAP were found among OZRs, OXR1-SCR and OXR1-SHR groups. On the 23 th day since virus was injected, the MAP in OXR1-SHR gradually decreased and significantly differed from other two groups on day 26; the weight gain were similar among groups, although body weight in OXR1-sh RNA was a littler lower compared to OZRs and OXR1-SCR;(2) Although the total power of heart variability not change, LF/HF ratio obviously decreased in OXR1-SHR group, compared with OZRs and OXR1-SCR;(3) OXR1 expression in PVN were inhibited in OXR1-SHR, and moreover, scrambled RNA did not influence OXR1 amount in PVN, compared with Con;(4) In vivo, functional studies found that SB334867 injected into PVN similarly decreased MAP and RSNA in OZRs and OXR1-SCR, but induced significant reductions in OXR1-sh RNA group. Orexin-induced pressor effects also were remarkably attenuated in OXR1-sh RNA;(5) The excitatory response of presympathetic neurons to orexin A was also significantly lowered.Summary: Collectively these data indicated that the OXR1-sh RNA efficiently downregulate OXR1 expression in PVN, which reversed hypertension in OZRs.Conclusions:Upregulation of orexin receptor 1 in PVN increased sympathetic tone output through over-exciting presympathetic neurons of PVN, thus causing elevation of heart rate and artery pressure. The neuroexciting effects of OXR1 activation related to increase of postsynaptic non-selective canton current. In contrast, downregulation of OXR1 expression in these sympathetic-regulated neurons in PVN of OZRs remarkably reduced neurons’ spontaneous activity, and furtherly rebalanced autonomic nerves system that finally reversed hypertension in OZRs.